LOCUS       sequence-232696-        4637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 24xSunTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4637)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 4637)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-21
FEATURES             Location/Qualifiers
     source          1..4637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             81..137
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             153..209
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             225..281
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             297..353
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             369..425
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             441..497
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             513..569
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             585..641
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             657..713
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             729..785
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             801..857
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             873..929
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             945..1001
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1017..1073
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1089..1145
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1161..1217
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1233..1289
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1305..1361
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1377..1433
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1449..1505
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1521..1577
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1593..1649
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             1737..1793
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     polyA_signal    1803..1924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1840..1859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1894..1913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1931..1964)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1971..2219
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1971..1991
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2142..2161
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2221..2254)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2291..2310)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2292..2310)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2397..2414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2568..3156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2648..2667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3327..4118,4119..4187))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3950..3969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4188..4292)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4360..4378
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4416..4438)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4538..4557
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gaagaacttt tgagcaagaa ttatcatctt gagaacgaag
      121 tggctcgtct taagaaaggt tctggcagtg gagaagaact gctttcaaag aattaccacc
      181 tggaaaatga ggtagctaga ctgaaaaagg ggagcggaag tggggaggag ttgctgagca
      241 aaaattatca tttggagaac gaagtagcac gactaaagaa agggtccgga tcgggtgagg
      301 agttactctc gaaaaattat catctcgaaa acgaagtggc tcggctaaaa aagggcagtg
      361 gttctggaga agagctatta tctaaaaact accacctcga aaatgaggtg gcacgcttaa
      421 aaaagggaag tggcagtggt gaagagctac tatccaagaa ttatcatctt gagaacgagg
      481 tagcgcgttt gaagaagggt tccggctcag gagaggaact gctctcgaag aactatcatc
      541 ttgaaaatga ggtcgctcga ttaaaaaagg gatcgggcag tggtgaggaa ctactttcaa
      601 agaattacca cctcgaaaac gaagtagctc gattaaagaa aggttcaggg tcgggtgaag
      661 aattactgag taaaaattat catctggaaa atgaggtagc gagactaaaa aaggggagtg
      721 gttctggcga agagttgcta tcgaaaaatt atcatcttga gaacgaagtt gctaggctca
      781 aaaagggctc aggctcaggc gaggagttgc tctcgaaaaa ctaccacttg gaaaatgagg
      841 tcgcgaggtt gaaaaagggg agcgggtcgg gcgaggagtt attgagcaaa aactatcatt
      901 tagagaacga agtcgcgcgc ttaaagaaag gctcgggctc gggcgaagaa ctcttatcga
      961 agaactacca cctcgaaaat gaggtcgcca ggttgaaaaa gggcagtggc agcggggagg
     1021 aactcttgag caagaactac cacttggaga atgaggtcgc gagattgaag aaagggtcgg
     1081 ggagcggcga ggaattgctc agcaagaatt atcatttgga gaacgaagtc gccaggctca
     1141 agaaaggctc ggggtcgggg gaggaattgt tgagtaaaaa ctaccacttg gaaaatgaag
     1201 tcgccaggct caaaaaaggg agtgggagcg gcgaagagtt attgagcaaa aattaccact
     1261 tggagaacga agtggcaagg ctcaagaaag ggagcggcag cggggaggag ctcttatcga
     1321 agaactacca cttagagaat gaagtcgccc gcttgaagaa aggctcgggg agcggggaag
     1381 agctcttgag caagaactac cacttggaaa atgaggtggc gcgcttgaag aaagggagcg
     1441 ggagcgggga agagttacta tctaagaatt atcatctcga gaacgaggtg gctcgactaa
     1501 agaagggctc cggcagtggg gaggaactcc tgtcgaagaa ctatcatctt gaaaatgagg
     1561 ttgcaagact taaaaagggg tccggatcag gtgaggaact actcagtaag aattaccacc
     1621 tggaaaacga agttgcacgt ttgaagaaag gatcaggatc aggcgaagaa ctgctctcaa
     1681 aagattatca tttggaaaat gaggttgcac gtttaaaaaa gggaagtggc agtggtgagg
     1741 aacttctgtc gaaaaattat catctcgaga atgaagtagc ccgacttaaa aagtgaacta
     1801 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1861 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1921 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgag gagggcctat
     1981 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     2041 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     2101 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     2161 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     2221 ataacttcgt atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat
     2281 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa
     2341 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2401 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2461 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2521 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2581 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2641 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2761 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2821 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2881 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2941 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3001 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3061 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3121 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3181 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3241 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3301 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3361 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3421 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3481 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3541 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3601 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3661 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3721 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3781 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3841 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3901 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3961 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4021 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4081 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4141 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4201 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4261 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4321 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     4381 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4441 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4501 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     4561 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta
     4621 tgtatcatac acatacg
//
